Antibodies binding to the extracellular domain of the receptor tyrosine kinase ALK

ABSTRACT

The present invention concerns an antibody specific for human ALK (Anaplastic Lymphoma Kinase), in particular a scFv, a nucleic acid sequence encoding it, its production and its use as a pharmaceutical or for diagnostic purposes. Said antibody is suitable for the local treatment of tumors, in particular glioblastoma.

RELATED INFORMATION

The application claims priority to U.S. provisional patent applicationNo. 60/795,831, filed on Apr. 28, 2006, the entire contents of which arehereby incorporated by reference.

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

TECHNICAL FIELD

The present invention concerns an antibody specific for human ALK(Anaplastic Lymphoma Kinase), in particular a scFv, a nucleic acidsequence encoding it, its production and its use as a pharmaceutical orfor diagnostic purposes. Said antibody is suitable for the localtreatment of tumors or cancer, in particular glioblastoma.

BACKGROUND ART

ALK (Anaplastic Lymphoma Kinase; CD246) is a member of the receptortyrosine kinase (RTK) family. As a typical member of this family, it isa type-I transmembrane protein essentially consisting of three domains:the extracellular ligand-binding domain (aa19-1038), which contains oneLDL-receptor class A domain and two MAM domains (MAM: Meprin, A5antigen, protein tyrosine phosphatase μ), a transmembrane domain(aa1039-1059) and a cytoplasmic domain (aa 1060-1620), containing thetyrosine kinase domain. A signal peptide is present at the N-terminus ofthe nascent protein (aa 1-18), which is cleaved upon secretion.

The full-length human and mouse ALK were cloned in 1997 by twoindependent groups (Iwahara 1997; Morris 1997). ALK is highly similar tothe RTK called Leukocyte Tyrosine Kinase (LTK) and belongs to theinsulin receptor superfamily. ALK exhibits 57% aa identity and 71% aasimilarity with LTK in their regions of overlap (Morris 2001). ALK ishighly N-glycosylated and contains 21 putative N-glycosylation sites.Amino acids 687 to 1034 have significant similarity (50% aa identity) toLTK. However, the N-terminus proximal 686 aa sequence shows no homologyto any known proteins with the exception of a very short sequence alsofound in the LDL receptor (Duyster 2001/SWISSPROT). In addition, itcontains two MAM domains at aa264-427 and aa478-636 (Meprin, A5 antigen,protein tyrosine phosphatase μ). These domains are thought to have anadhesive function, as they are widespread among various adhesiveproteins implicated in cell-to-cell interaction (De Juan 2002).Furthermore, there is a binding site for the ALK putative ligandscorresponding to amino acids 396-406 (Stoica 2001; see below). The aminoacid sequence of the kinase domain of murine ALK shows 98% aa-identityto human ALK, 78% identity to mouse LTK, 52% to mouse ros, 47% to humaninsulin-like growth factor receptor and 46% to human insulin receptor(Iwahara 1997; Ladanyi 2000). No splice variants of ALK have beendescribed to date. However, ALK is often associated with chromosomaltranslocations (see below).

The ALK gene spans about 315 kb and has 26 exons. Much of the geneconsists of two large introns that span about 170 kb. The ALK transcriptis 6.5 kb of length (Kutok 2002). According to Morris, the cDNA spans6226 bp (Morris 2001).

ALK expression in mice starts during embryogenesis around thedevelopment stage E11 and is persisting in the neonatal periods ofdevelopment where it is expressed in the nervous system. In the adult,its physiological expression is restricted to certain neuronal (neuraland glial cells and probably endothelial cells) regions of the CNS atlow levels (Morris 1997; Duyster 2001; Stoica 2001). Actually, theabundance of ALK decreases in the postnatal period (Morris 2001). Basedon its expression pattern, a role for the receptor in brain developmentis suggested (Duyster 2001). The neural-restricted expression of ALKsuggests that it serves as a receptor for neurotrophic factors (seelater). Consistent with this, its expression pattern overlaps with thegenes encoding the TRK family of neurotrophin receptors (Morris 2001).However, ALK knockout mice do not show any obvious phenotype(unpublished data), which might be due to some functional redundancywith TRK family members or other neurotrophin receptors. Notably,hematopoietic tissues show no detectable expression of ALK (see Morris2001).

Two potential ligands for ALK have recently been described,“pleiotrophin” (PTN) and “midkine” (MK) (Stoica 2001; Duyster 2001;Stoica 2002). The PTN-ALK interaction was identified by using purifiedhuman pleiotrophin protein to screen a phage display peptide library. Bythis method, a sequence of ALK present in its extracellular domain (aa396-406) was identified. Importantly, this sequence is not shared withLTK, the RTK most closely related to ALK. This ligand-binding region isalso conserved in the potential homologue of ALK in Drosophila (Loren2001). ALK is phosphorylated rapidly upon PTN binding (Bowden 2002).Moreover, ALK has been shown to be stimulated by pleiotrophin in cellculture. This makes the pleiotrophin/ALK interaction particularlyinteresting in the light of the pathological implications pleiotrophinhas (Stoica 2001). Cell lines that lack ALK expression also fail to showa growth response to pleiotrophin and vice versa (Stoica 2001). In vivo,elevated pleiotrophin levels in the serum of patients suffering fromvarious solid tumors have been demonstrated, and animal studies havesuggested a contribution of pleiotrophin to tumor growth (Stoica 2001).The role of PTN as rate-limiting angiogenic factor in tumor growth iswell established in animal models (Choudhuri 1997). In 1996 Czubayko etal. demonstrated the importance of PTN in tumor angiogenesis, inprevention of apoptosis and metastasis by modulating PTN levels with aribozyme targeting approach (Czubayko 1996). Serum level measurements ofPTN in mice demonstrated a clear correlation with the size of the tumor.PTN plays a significant role in some of the most aggressive human cancertypes such as melanoma and pancreatic cancer thus giving interestingperspectives for potential further applications of an ALK inhibitor(Weber 2000; Stoica 2001). In human patients, elevated serumpleiotrophin levels were found in patients with pancreatic cancer (n=41;P<0.0001) and colon cancer (n=65; P=0.0079). In healthy individuals, PTNis expressed in a tightly regulated manner during perinatal organdevelopment and in selective populations of neurons and glia in theadult.

Co-expression of PTN and ALK, as found in several cancer cell lines,indicates that they could form an autocrine loop of growth stimulation(Stoica 2001). In spite of all these data, the literature indicates thatis not yet clear if the effects of PTN are mediated by ALK alone and/orby other unidentified PTN receptors (Duyster 2001). At least two otherpotential receptors of PTN have been suggested: the receptor tyrosinephosphatase RPTPβ and the heparan sulfate proteoglycan N-syndecan.However, RPTPβ might act as a signalling modulator of PTN/ALK signallingand N-syndecan as a chaperone for the ligand (Bowden 2002).

Recently, another secreted growth factor related to pleiotrophin calledmidkine (MK) has been identified as a second ligand for ALK. Similarlyto PTN, binding and activating functions (e.g. induction of soft agarcolony formation in cell cultures) of MK can be blocked by the sameantibody raised against the ALK-ECD (Stoica 2001). Like pleiotrophin,midkine is upregulated in many tumors, although its physiologicalexpression is very restricted in adult normal tissues (Stoica 2002).Analysis of 47 bladder tumor samples revealed that MK expression issignificantly (about four times) enhanced as compared to normal bladdertissue. Furthermore, pronounced overexpression correlates with poorpatient survival (O'Brien 1996).

However, the affinity of MK for ALK is about 5 times lower than the oneof pleiotrophin (Stoica 2002). Interestingly, as with pleiotrophin,inhibition of ALK via ribozymes also inhibits the effects of MK in cellculture (Stoica 2002). The authors of these studies also come to theconclusion that inhibition of the PTK/MK/ALK pathway opens veryattractive possibilities for the treatment of various diseases, some ofthem having very limited treatment options so far, such as, for example,glioblastoma and pancreatic cancer. (Stoica 2002).

In healthy individuals, ALK mRNA expression peaks during the neonatalperiod and persists in adults in a few selected portions of the nervoussystem. Recently, expression of the ALK protein was also detected inendothelial cells that were associated to neuronal and glial cells.Evidence that at least a part of the malignant activities described forpleiotrophin are mediated through ALK came from experiments in which theexpression of ALK was depleted by a ribozyme targeting approach. Suchdepletion of ALK prevented pleiotrophin-stimulated phosphorylation ofthe anti-apoptopic protein Akt and led to a prolonged survival of micethat had received xenografts. Indeed, the number of apoptopic cells inthe tumor grafts was significantly increased, when ALK expression wasdepleted (Powers 2002).

Evidence that malignant activities described for MK are mediated throughALK came from experiments with monoclonal antibodies directed againstthe ALK ECD. Addition of a 1:25 dilution of hybridoma cell supernatantfrom two anti-ALK ECD antibodies leads to a significant decrease incolony formation of SW-13 cells in soft agar (Stoica 2002). Analysis often different cell lines revealed that the ability for a growth responseto PTN perfectly correlated with the expression of ALK mRNA (thefollowing cell lines responded to PTN and were found to express ALKmRNA: HUVEC, NIH3T3, SW-13, Colo357, ME-180, U87, MD-MB 231; Stoica2001). Interestingly, in some cancer cell lines (Colo357 pancreaticcancer, Hs578T breast cancer and U87 glioblastoma), PTN and ALK areco-expressed, indicating that PTN and ALK form an autocrine loop ofgrowth stimulation (Stoica 2001).

Interestingly, both PTN and MK have been shown to cause transcriptionalup-regulation of the anti-apoptotic bcl-2 protein (Stoica 2002). Inaddition activated Akt (which is a crucial downstream target of aberrantALK signalling) phosphorylates the pro-apoptotic factor called bad, thusleading to dissociation from bclxl, which, when liberated from bad, cansuppress apoptosis by blocking the release of cytochrome c (see Bowden2002 for references).

Aberrant expression of ALK might be involved in the development ofseveral cancers. However, it was first associated with a subgroup ofhigh-malignant Non-Hodgkin lymphomas (NHLs), the so-called AnaplasticLarge Cell Lymphomas (ALCLs). Non-Hodgkin lymphomas represent clonalneoplasias originating from various cells of lymphatic origin.

Most patients with the primary systemic clinical subtype of ALCL havethe t2,5 translocation, expressing a fusion protein that joins theN-terminus of nucleophosmin (NPM) to the C-terminus of ALK. The fusionconsists of aa 1-117 of NPM fused to aa 1058-1620 of ALK and thechromosomal breakage is located in an intron located between the exonsencoding the TM and juxtamembrane domain of ALK (Duyster 2001). NPM-ALKis a transcript containing an ORF of 2040 bp encoding a 680aa protein(Morris 2001). This corresponds to a breakage in intron 4 of NPM, whichspans 911 bp and intron 16 of ALK which spans 2094 bp (Kutok 2002). Mostlikely the ALK sequence in this fusion protein is the minimal sequencerequired for the protein to lead to ALCL (Duyster 2001). The inversefusion (ALK-NPM) is not expressed, at least not in lymphoid cells (Kutok2002). The wild-type NPM protein demonstrates ubiquitous expression andfunctions as a carrier of proteins from the cytoplasm into thenucleolus. As a matter of fact, NPM is a 38 kDa nuclear protein encodedon chromosome 5 that contains a NLS, binds nuclear proteins and engagesin cytoplasm/nuclear trafficking (Duyster 2001). NPM is one of the mostabundant nucleolar proteins and is normally present as a hexamer (Morris2001). Most importantly NPM normally undergoes self-oligomerization(hexamers) as well as hetero-oligomerization with NPM-ALK (Duyster2001). The 2;5 translocation brings the ALK gene portion encoding thetyrosine kinase on chromosome 2 under the control of the strong NPMpromoter on chromosome 5, producing permanent expression of the chimericNPM-ALK protein (p80) (Duyster 2001). Hence, ALK kinase is deregulatedand ectopic, both in terms of cell type (lymphoid) and cellularcompartment (nucleus/nucleolus and cytoplasm) (Ladanyi 2000). Thelocalization (cytoplasm or nucleus) of NPM seems not to affect itseffect on lymphomagenesis (Duyster 2001). The resultant aberranttyrosine kinase activity triggers malignant transformation viaconstitutive phosphorylation of intracellular targets. Various otherless common ALK fusion proteins are associated with ALCL. All variantsdemonstrate linkage of the ALK tyrosine kinase domain to an alternativepromoter that regulates its expression.

Full-length ALK has been reported to be also expressed in about 92% ofprimary neuroblastoma cells and in some rhabdomyosarcomas (Lamant 2000).However, no correlation between ALK expression and tumor biology hasbeen demonstrated so far. This fact, taken together with the lack ofevidence regarding significant levels of endogenously phosphorylated ALKin these tumors, suggest that ALK expression in neuroblastoma reflectsits normal expression in immature neural cells rather than a primaryoncogenic role and ALK in these tumors is not constitutivelyphosphorylated thus questioning an important role for ALK in thesetumors (Duyster 2001; Pulford 2001). Nevertheless, ALK signalling mightbe important in at least some neuroblastomas, as suggested by Miyake etal., who found overexpression and constitutive phosphorylation of ALKdue to gene amplification in neuroblastoma-derived cell lines (Miyake2002). However, other neuroblastoma-derived cell lines do not showconstitutive activation of ALK, thus arguing against a generalpathological involvement of ALK (Dirks 2002; Pulford 2004).

Most interestingly, ALK seems to be important for growth of glioblastomamultiforme, a highly malignant brain tumor that offers very limitedtherapeutic options (Powers 2002). Multiple genetic alterations havebeen shown to occur in these devastating tumors including loss ormutations of PTEN, p53 and INK4a-ARF. In addition, RTK signalling playsa particularly important role in growth and development of these tumors,which overexpress various growth factors such as PDGF, HGF, NGF and VEGFsuggesting autocrine RTK signalling loops. Powers and colleagues haveshown mRNA and protein expression of ALK in glioblastoma patient tumorsamples, whereas the signals were not detectable in normal adjacentbrain tissue (Powers 2002). Furthermore, human U87MG glioblastoma cells(which are derived from a patient and represent a well-characterizedmodel system to study tumorigenesis and signalling in glioblastoma) showALK-dependent anti-apoptotic behaviour in xenograft studies. When ALK isdepleted in these tumor cells by the use of ribozymes, mice injectedwith these tumor cells survive at least twice as long as when injectedwith wild-type tumor cells, and these tumor cells show drasticallyincreased apoptosis. Thus, ALK and its ligand(s) provide an essentialsurvival signal that is rate-limiting for tumor growth of U87MG cells invivo (Powers 2002). These finding indicate that inhibition of ALKsignalling could be a promising approach to improve life expectancy ofglioblastoma patients.

Glioblastoma multiforme is by far the most common and malignant primaryglial tumor with an incidence of about 2/100'000/y (about 15'000 casesin US and Western Europe per year). It affects preferentially thecerebral hemispheres, but can also affect the brain stem (mainly inchildren) or the spinal cord. The tumors may manifest de novo (primaryglioblastoma) or may develop from lower grade astrocytomas (secondaryglioblastoma). Primary and secondary glioblastomas show little molecularoverlap and constitute different disease entities on molecular level.They both contain many genetic abnormalities including affection of p53,EGFR, MDM2, PDGF, PTEN, p16, RB.

No significant therapy advancement has occurred in the last 25 years.Therapies are only palliative and can expand the life expectance from 3months to 1 year. Patients usually present with slowly progressiveneurological deficit, e.g. motor weakness, intracranial pressuresymptoms, e.g. headache, nausea, vomiting, cognitive impairment, orseizures. Changes in personality can also be early signs. The etiologyof glioblastoma is unknown, familial cases represent less than 1%. Theonly consistent risk factor identified is exposure to petrochemicals.Diagnosis is made mainly by imaging studies (CT, NMR) and biopsy.Completely staging most glioblastomas is neither practical nor possiblebecause these tumors do not have clearly defined margins. Rather theyexhibit well-known tendencies to invade locally and spread along compactwhite matter pathways. The primary reason why no curative treatment ispossible is because the tumor is beyond the reach of local control whendiagnosed. The primary chemotherapeutic agents are carmustine (analkylating agent) and cisplatinum but only 40% of patients show someresponse.

Although there are quite some uncertainties regarding the role of ALK inglioblastoma, this disease offers various approaches for ALK-directeddrugs. In fact, for this devastating disease even a small improvement ofcurrent therapy options would serve an enormous medical need. It isimportant to note that since glioblastoma cells express the full-lengthALK, for treating this cancer ALK could be considered as a target notonly for small molecule kinase inhibitors but also for antibodies and/orantibody fragments such as scFvs i.e. to induce apoptosis of tumorcells. The strict localization of glioblastoma to the CNS supports theuse of scFvs, if they can be delivered efficiently to the CNS (no rapidclearance due to compartmentalization, but better tumor penetrationcompared to IgGs due to their smaller size). Antibodies and/or antibodyfragments could be directed against the ligand-binding sequence of ALK(aa 396-406) or against other parts of the extracellular parts of thereceptor.

The very limited expression of ALK in healthy tissues underphysiological conditions indicates that tumors expressing ALK might bean excellent target for disease treatment using radioactive ortoxin-labelled antibodies and/or antibody fragments, irrespective ofwhether ALK is involved in the pathogenesis of these tumors or not. Inaddition to glioblastoma cells, ALK expression has been found with highsignificance in melanoma cell lines and breast carcinoma cell lines(without being constitutively phosphorylated) (Dirks 2002). The factthat a large portion of the extracellular domain of ALK seems to berather unique in the human proteome should make this approach highlyspecific.

WO9515331/U.S. Pat. No. 5,529,925 discloses the cloning and sequencingof the human nucleic acid sequences, which are rearranged in the t(2;5)(p23; q35) chromosomal translocation event which occurs in human t(2;5) lymphoma. The rearrangement was found to bring sequences from thenucleolar phosphoprotein gene (the NPM gene) on chromosome 5q35 to thosefrom a previously unidentified protein tyrosine kinase gene (hereinafterthe ALK gene) on chromosome 2p23. The sequence of the fusion gene andfusion protein (NPM/ALK fusion gene or protein, respectively) were alsodisclosed.

The full-length ALK sequence is patented in U.S. Pat. No. 5,770,421,entitled “Human ALK Protein Tyrosine Kinase.” Furthermore, the patentU.S. Pat. No. 6,174,674B1 entitled “Method of detecting a chromosomalrearrangement involving a breakpoint in the ALK or NPM gene”, disclosesprimers for detecting the NPM-ALK fusion sequence in patient samples. Inanother patent, U.S. Pat. No. 6,696,548 entitled “ALK protein tyrosinekinase/receptor and ligands thereof”, the use of ALK for detection ofALK ligands and antibodies binding to specific sequences of ALK isdisclosed. It also discloses a method of identifying an agent capable ofbinding to the isolated ALK polypeptide. WO0196394/US20020034768discloses ALK as receptor of pleiotrophin. US20040234519 disclosesanti-pleiotrophin antibodies, and WO2006020684 describes the detectionof pleitrophin.

DISCLOSURE OF THE INVENTION

Hence, it is a general object of the invention to provide a stable andsoluble antibody or antibody derivative, which binds the human ALKprotein in vitro and in vivo. Most preferably, the antibody isspecifically targeted against the ligand-binding domain of ALK (aminoacids 396-406) and hence will block both the biologic effects of MK,which has a Kd for ALK of about 170 pM, as well as the biologic effectsof PTN, which has a Kd for ALK of about 20-30 pM (Stoica 2002; Stoica2001). In a preferred embodiment said antibody or antibody fragment is ascFv antibody or a Fab fragment. In the following the term antibodycomprises full-length antibodies as well as other antibody derivatives.

Now, in order to implement these and still further objects of theinvention, which will become more readily apparent as the descriptionproceeds, said antibody is manifested by the features that it comprisesa variable heavy chain CDR3 of a sequence of at least 50% identity tothe sequence SEQ. ID. No. 2. Preferably, the sequence identity is atleast 60%, 65%, 75%, 85%, or more preferably at least 92%. Mostpreferably, said antibody has a VH CDR3 of the sequence SEQ. ID. No. 2.

In one embodiment, the antibody or antigen binding portion thereof ofthe invention specifically binds to a particular epitope of the ALKprotein. Such epitopes reside, for example, within amino acids 1-50,50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450,450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-900,900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, or1500-1620 of the ALK protein, or any interval, portion or range thereof.In one embodiment, the antibody or antigen binding portion thereofspecifically binds to an epitope comprising, essentially consisting ofor a fragment of the region spanning amino acid residues 391±3 and 406±3(SEQ. ID No: 91 shows amino acid residues 388 to 409 of the human ALKprotein), preferably amino acids 391-406 (SEQ. ID. NO: 1) of the ALKprotein (SEQ ID NO: 1). It is understood that the indicated range is notto be considered as having sharp boundaries, but that the antibody orantigen binding portion thereof may bind or partially bind in a regionclosely situated to or within the ligand-binding domain of ALK.Preferably, the antibodies or antibody-derivatives bind to an epitope ofthe ALK protein of 10 to 20 amino acids in length.

In another embodiment the antibody or antigen binding portion thereofcan be characterized as specifically binding to an ALK protein with aK_(D) of less than about 10×10⁻⁶ M. In a particular embodiment, theantibody or antigen binding portion thereof specifically binds to an ALKprotein (or fragment thereof) with a K_(D) of at least about 10×10⁻⁷ M,at least about 10×10⁻⁸ M, at least about 10×10⁻⁹ M, at least about10×10⁻¹⁰ M, at least about 10×10⁻¹¹ M, or at least about 10×10⁻¹² M or aK_(D) even more favorable.

In various other embodiments, the antibody or antigen binding portionthereof includes a variable heavy chain region comprising an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, or more preferably at least99% identical to a variable heavy chain region amino acid sequence asset forth in SEQ ID NO: 4.

In other embodiments, the antibody or antigen binding portion thereofincludes a variable light chain region comprising an amino acid sequenceat least 80%, 85%, 90%, 95%, 98% or more preferably at least 99%identical to a variable light chain region amino acid sequence as setforth in SEQ ID NO: 5.

In still other embodiments, the antibody or antigen binding portionthereof includes both a variable heavy chain region comprising an aminoacid sequence at least 80%, 85%, 90%, 95%, 98% or more preferably atleast 99% identical to a variable heavy chain region amino acid sequenceas set forth in SEQ ID NO: 4 and a variable light chain regioncomprising an amino acid sequence at least 80%, 85%, 90%, 95%, 98% ormore preferably at least 99% identical to a variable light chain aminoacid sequence as set forth in SEQ ID NO: 5.

In certain other embodiments, the antibody or antigen binding portionthereof specifically bind to an epitope that overlaps with an epitopebound by an antibody or antibody derivative of ESBA521 (Seq. ID. No. 19)and/or competes for binding to an ALK protein, or portion thereof, withan antibody or antibody derivative of ESBA521. In a related embodiment,the antibody or antigen binding portion thereof specifically binds to anepitope comprising residues 391-406 (SEQ ID NO: 1) of an ALK protein, orportion thereof.

The variable heavy and light chain regions of the antibodies or antigenbinding portions thereof typically include one or more complementaritydetermining regions (CDRs). These include the CDR1, CDR2, and CDR3regions. In particular embodiments, the variable heavy chain CDRs are atleast 80%, 85%, 90%, 95%, or more preferably 100% identical to a CDR ofthe ESBA521 antibody. In other particular embodiments, variable lightchain CDRs are at least 80%, 85%, 90%, 95%, or more preferably 100%,identical to a CDR of a variable light chain region of the ESBA521antibody.

Accordingly, particular antibodies or fragments of the inventioncomprise a variable heavy chain region that includes one or morecomplementarity determining regions (CDRs) that are at least 80%, 85%,90%, 95%, or more preferably 100%, identical to a CDR of a variableheavy chain region of the ESBA521 and a variable light chain region thatincludes one or more CDRs that are at least 80%, 85%, 90%, 95% or morepreferably 100%, identical to a CDR of a variable light chain region ofthe ESBA521 antibody.

The variable heavy chain region of the antibodies or antigen bindingportions thereof can also include all three CDRs that are at least 80%,85%, 90%, 95%, or more preferably 100%, identical to the CDRs of thevariable heavy chain region of the ESBA521 antibody and/or all threeCDRs that are at least 80%, 85%, 90%, 95% or more preferably 100%,identical to the CDRs of the variable light chain region of the ESBA521antibody.

In another embodiment of the invention, the antibodies or antigenbinding portions thereof (a) include a heavy chain variable region thatis encoded by or derived from (i.e. is the product of) a human VH gene(e.g., H3 type); and/or (b) include a light chain variable region thatis encoded by or derived from a human V kappa or lambda gene (e.g.,lambda1 type).

The antibodies of the present invention include full-length antibodies,for example, monoclonal antibodies, that include an effector domain,(e.g., an Fc domain), as well as antibody portions or fragments, such assingle-chain antibodies and Fab fragments. The antibodies can also belinked to a variety of therapeutic agents (e.g., anticancer agents,chemotherapeutics, or toxins) and/or a label (e.g., radiolabel).

In another aspect, the invention features isolated nucleic acidsincluding a sequence encoding an antibody heavy chain variable regionwhich is at least 75%, 80%, 85%, 90%, 95%, or more preferably at least99%, identical to SEQ ID NO: 22. The invention also features isolatednucleic acids that include a sequence encoding an antibody light chainvariable region which is at least 75%, 80%, 85%, 90%, 95%, or morepreferably at least 99%, identical to SEQ ID NO: 21.

The invention also features expression vectors including any of theforegoing nucleic acids either alone or in combination (e.g., expressedfrom one or more vectors), as well as host cells comprising suchexpression vectors.

Suitable host cells for expressing antibodies of the invention include avariety of eukaryotic cells, e.g., yeast cells, mammalian cells, e.g.,Chinese hamster ovary (CHO) cells, NS0 cells, myeloma cells, or plantcells. The molecules of the invention can also be expressed inprokaryotic cells, e.g., E. coli.

The invention also features methods for making the antibodies or antigenbinding portions thereof by expressing nucleic acids encoding antibodiesin a host cell (e.g., nucleic acids encoding the antigen binding regionportion of an antibody). In yet another aspect, the invention features ahybridoma or transfectoma including the aforementioned nucleic acids.

In another embodiment, the invention provides an antigen comprising anepitope of the ALK protein, preferably of the PTN ligand binding domain,more preferably a fragment comprising, essentially consisting of or afragment of the region spanning amino acid residues 391±3 and 406±3 (seeSEQ. ID No: 91 which shows amino acid residues 388 to 409 of the humanALK protein), most preferably amino acids 391-406 (SEQ. ID. No: 1). Theantigen can be used for raising, screening, or detecting the presence ofan anti-ALK antibody or can be used as an agent in active immunotherapy,i.e. as a vaccine.

As a vaccine, the antigen can be used alone or in combination with anappropriate adjuvant or hapten, e.g., mixed or conjugated eitherchemically or genetically. The antigen when used for activeimmunotherapy can also be used in combination with passiveimmunotherapy, for example, with any of the anti-ALK antibodiesdisclosed herein, or in combination with a monoclonal or polyclonalpreparation of anti-ALK antibodies, e.g., serum gammaglobulin from aseropositive donor.

In another embodiment, the antibody molecules (or VL and VH bindingregions) are fully human. Treatment of humans with human monoclonalantibodies offers several advantages. For example, the antibodies arelikely to be less immunogenic in humans than non-human antibodies. Thetherapy is also rapid because ALK inactivation can occur as soon as theantibody reaches a cancer site (where ALK is expressed). Therefore, in arelated embodiment, the antibody is a scFv antibody, i.e., ESBA521 or anantibody comprising a VL and/or VH region(s) (or CDRs thereof; e.g., VLCDR3 (SEQ ID NO:3) and/or VH CDR3 (SEQ ID NO: 2)) of ESBA521.

Human antibodies also localize to appropriate sites in humans moreefficiently than non-human antibodies. Furthermore, the treatment isspecific for ALK, is recombinant and highly purified and, unliketraditional therapies, avoids the potential of being contaminated withadventitious agents. Alternatively, antibodies and antibody-derivativesof the present invention may be produced by chemical synthesis.

In another embodiment, the invention provides compositions for treatinga cancer (or the making of a medicament so suited) that can preventneoplasia in a subject by competing with ligands of ALK such as midkine(MK) and/or pleiotrophin (PTN) and thereby block ALK-signaling mediatedby such ligands. Such a composition can be administered alone or incombination with art recognized anti-cancer agents, for example,methotrexate, and the like.

The antibody of the invention and/or ALK vaccine can be used alone or incombined with a known therapeutic, e.g., an anti-cancer agent, e.g.,methotrexate and the like.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood and objects other than those setforth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings, wherein:

FIG. 1 shows a scheme of the human ALK protein used. A 16 amino acidpeptide of the PTN binding site (dashed) is used as the epitope in a twohybrid screen for scFv binders.

FIG. 2 shows the stepwise randomization of VH CDR3, VL CDR 3 and VH CDR2 portions to obtain ESBA521 as secondary binder and a set of scFvs astertiary binders (see Tab. 1). X stands for any amino acid residue.

FIG. 3 shows an ELISA experiment wherein the binding characteristics ofimproved scFvs are compared to that of the framework they originatefrom.

FIG. 4 shows immunostaining of transiently transfected HeLa cells withESBA521 (left panels) and a polyclonal ALK specific antibody (rightpanels). Middle panel: same cells visualized by light microscopy.

FIG. 5 shows an ELISA experiment comparing the ESBA512 to the improvedtertiary binders.

DETAILED DESCRIPTION OF THE INVENTION

In order that the present invention may be more readily understood,certain terms are first defined.

DEFINITIONS

The term “ALK” and “Alk-1” includes the human ALK protein encoded by theALK (Anaplastic Lymphoma Kinase) gene which is a membrane-spanningprotein tyrosine kinase (PTK)/receptor.

The term “antibody” refers to whole antibodies and any antigen bindingfragment (i.e., “antigen-binding portion,” “antigen bindingpolypeptide,” or “immunobinder”) or single chain thereof. An “antibody”refers to a glycoprotein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, or an antigenbinding portion thereof. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as V_(H)) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”) refer to one or more fragments of an antibody that retain theability to specifically bind to an antigen (e.g., ALK). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and CH1domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains ofa single arm of an antibody, (v) a single domain or dAb fragment (Wardet al., (1989) Nature 341:544-546), which consists of a V_(H) domain;and (vi) an isolated complementarity determining region (CDR) or (vii) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Antigen-binding portions can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins. Antibodies can be of different isotype, forexample, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1,IgA2, IgD, IgE, or IgM antibody.

The term “frameworks” refers to the art recognized portions of anantibody variable region that exist between the more divergent CDRregions. Such framework regions are typically referred to as frameworks1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for holding,in three-dimensional space, the three CDRs found in a heavy or lightchain antibody variable region, such that the CDRs can form anantigen-binding surface. Such frameworks can also be referred to asscaffolds as they provide support for the presentation of the moredivergent CDRs. Other CDRs and frameworks of the immunoglobulinsuperfamily, such as ankyrin repeats and fibronectin, can be used asantigen binding molecules (see also, for example, U.S. Pat. Nos.6,300,064, 6,815,540 and U.S. Pub. No. 20040132028).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds (e.g.,ALK, for example, amino acid residues 391-406 of human ALK-1 (see e.g.,SEQ ID NO: 1). An epitope typically includes at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to antibody binding to an epitope on apredetermined antigen. Typically, the antibody binds with an affinity(K_(D)) of approximately less than 10⁻⁷ M, such as approximately lessthan 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

The term “K_(D),” refers to the dissociation equilibrium constant of aparticular antibody-antigen interaction. Typically, the antibodies ofthe invention bind to ALK with a dissociation equilibrium constant(K_(D)) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument.

The terms “neutralizes ALK,” “inhibits ALK,” and “blocks ALK” are usedinterchangeably to refer to the ability of an antibody of the inventionto prevent ALK from interacting with one or more target ligands and, forexample, triggering signal transduction.

The term “nucleic acid molecule,” refers to DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. A nucleic acidis “operably linked” when it is placed into a functional relationshipwith another nucleic acid sequence. For instance, a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand. Such hybridization conditions are know in theart, and described, e.g., in Sambrook et al. infra.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weightof 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide oramino acid sequences can also be determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package, using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in the SEQ ID NOs of thepresent invention, i.e., nucleotide and amino acid sequencemodifications which do not abrogate the binding of the antibody encodedby the nucleotide sequence or containing the amino acid sequence, to theantigen. Such conservative sequence modifications include nucleotide andamino acid substitutions, additions and deletions. For example,modifications can be introduced by standard techniques known in the art,such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-ALK antibodyis preferably replaced with another amino acid residue from the sameside chain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997))

Alternatively, in another embodiment, mutations are randomly introducedalong all or part of an anti-ALK antibody coding sequence, such as bysaturation mutagenesis, and the resulting modified anti-ALK antibodiescan be screened for binding activity. A “consensus sequence” is asequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In afamily of proteins, each position in the consensus sequence is occupiedby the amino acid occurring most frequently at that position in thefamily. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

By reference to the tables and figures provided herein a consensussequence for the antibody heavy/light chain variable region CDR(s) canbe derived by optimal alignment of the amino acid sequences of thevariable region CDRs of the antibodies which are reactive againstepitope 390-406 of the human ALK-1 protein.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.

The term “host cell” refers to a cell into which and expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria, which are susceptible to transformation,include members of the enterobacteriaceae, such as strains ofEscherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis;Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitablemicrobes include Saccharomyces cerevisiae and Pichia pastoris. Suitableanimal host cell lines include CHO (Chinese Hamster Ovary lines) and NS0cells.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, anantibody of the present invention, for example, a subject having anALK-mediated disorder or a subject who ultimately may acquire such adisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disorder or recurring disorder,or in order to prolong the survival of a subject beyond that expected inthe absence of such treatment.

The term “ALK-mediated disorder” refers to disease states and/orsymptoms associated with ALK-mediated cancers or tumors. In general, theterm “ALK-mediated disorder” refers to any disorder, the onset,progression or the persistence of the symptoms of which requires theparticipation of ALK. Exemplary ALK-mediated disorders include, but arenot limited to, for example, cancer, in particular, glioblastoma.

The term “effective dose” or “effective dosage” refers to an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “subject” refers to any human or non-human animal. For example,the methods and compositions of the present invention can be used totreat a subject with a cancer, e.g., glioblastoma.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

The present invention provides in a first aspect an antibody binding thehuman ALK protein, said antibody comprising a variable heavy chain CDR3of a sequence with at least 50% sequence identity to the sequence SEQ.ID. No. 2. Preferably, the sequence identity is at least 60%, 70%, 75%,80%, or more preferably at least 90%. Most preferably, the CDR3 has theprecise sequence of SEQ. ID. No. 2.

In a preferred embodiment of the present invention the antibody bindsspecifically to the human ALK protein, i.e., it does not bind to themouse ALK protein, whose PTN binding site differs in only 2 amino acidresidues compared to the PTN binding site (SEQ. ID. No. 1) of the humanALK protein. The human isoleucine at position 3 is a valine and theaspartate is an alanine in the corresponding mouse sequence.

The antibody of the present invention can be a full-length antibody, butalso an antibody fragment, such as, for example, a scFv or a Fabfragment. Antigen binding fragments are well known in the art.Preferably, a scFv antibody is used.

The heavy chain and the light chain are composed of framework sequences,each comprising three CDRs, CDR1, CDR2 and CDR3, which are predominantlyinvolved in antigen binding. The antibody of the present inventioncomprises the VH domain of the H3 type and a VL domain of the lambda1type.

The VH and VL framework of the antibody of the present invention arestable and soluble so as to be functional in an intracellular reducingenvironment. Preferably it is the framework 4.4 that has previously beenisolated by a yeast screening system referred to as the “Quality controlsystem” (Auf der Maur et al., 2001; Auf der Maur et al. 2004). Thesequence of the framework can be deduced for example from SEQ. ID. No.20 (see below), where the framework portions are represented bynon-underlined and straight letters, while the CDR sequences areunderlined and the linker sequence is in italics.

The antibody of the present invention is able to bind a 16-amino acidALK epitope peptide of a sequence that is at least 75%, preferably 80%,85%, 90%, 95%, or most preferably 100%, identical to the sequence ofSEQ. ID. No. 1. This sequence is also referred to as PTN binding siteand is a unique sequence in the entire human genome. The correspondingmouse sequence varies in 2 out of 16 amino acids, i.e., V at position 3and A at position 7 instead of I or D, respectively. Preferably, theantibody of the present invention binds human but not mouse ALK, i.e.,is specific for the human protein. The ALK epitope comprising aboutresidues 391-406 or consisting of these residues is uniquely suited forselecting an antibody or antigen binding fragment that can specificallybind ALK and block or inhibit ALK-mediated activity. This epitope isalso suitable for screening or raising antibodies that specificallyblock ALK activity. Thus, this epitope, especially an epitope comprisingabout residues 391-406 or consisting of about these residues, isuniquely suited for use as an active immunotherapeutic agent or vaccineas further described herein.

The antibody of the present invention has an affinity for the ALKepitope peptide with a K_(d) of 30 nM or less, preferably 10 nM or less,most preferably below 3 nM.

In another embodiment of the present invention, the antibody comprisinga variable light chain CDR3 of a sequence with at least 50% sequenceidentity to the sequence SEQ. ID. No. 3. Preferably, the sequenceidentity is at least 60%, 70%, 80%, 85%, more preferably at least 90%.Most preferably, CDR3 is identical to SEQ. ID. No. 3. Again, thisantibody binds a 16-amino-acid ALK epitope peptide of a sequence with atleast 75%, preferably 80%, 85%, 90%, 95%, or most preferably 100%, aminoacid identity to the sequence SEQ. ID. No. 1. Also, the antibody has anaffinity for the ALK epitope peptide with a K_(d) Of less than 10 nM,preferably less than 7 nM.

In a preferred embodiment of the present invention the antibodycomprises a VH sequence of SEQ. ID. No. 4 and a VL sequence of SEQ. ID.No. 5. Additionally, it can comprise at least one mutation in at leastone of the CDRs resulting in a higher affinity characterized by a K_(d)Of less than about 3 nM. Said at least one mutation is preferably inCDR1 or CDR2 of VH and/or VL, most preferably in CDR2 of VH.

In another preferred embodiment of the present invention the antibodycomprises a variable heavy chain CDR2 comprising a sequence selectedfrom the group of SEQ. ID. No. 7, SEQ. ID. No. 8, SEQ. ID. No. 9, SEQ.ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 12, or SEQ. ID. No. 13.Preferably these defined CDR2 sequences are preceded by the amino acidsresidues AI and followed by the sequences of SEQ. ID. No. 17, so thatthe entire CDR2 is defined.

A preferred antibody of the present invention comprises a VH sequence ofSEQ. ID. No. 4 and a VL sequence of SEQ. ID. No. 5. In scFv antibodies,the domain structure can NH₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH;preferably, the linker has the sequence SEQ. ID. No. 16. Alternatively,the variable regions represented by SEQ ID NOS: 4 and 5 can beengineered into a full length antibody, e.g., IgG or IgM. Constantregions suitable for combining with the variable regions of theinvention are known in the art.

Within the scope of the present invention is the use of the antibody orantibody derivative as a medicament or as a diagnostic tool. Preferably,the production of a medicament for the treatment of cancers or tumors isenvisaged. For this purpose an antibody can be radiolabelled usingradionuclides or radiometal labeling. This is particularly valuable fortumor targeting, imaging and biodistribution studies. Also, recombinantDNA technology makes it possible to genetically fuse coding regions ofvariable V genes to modified toxin domains. For example, a scFv-toxinfusion wherein the scFv is specific for a tumor marker protein cantarget the toxin to the tumor, where the toxin causes cytotoxicity. Suchtargeted therapy results in the selective concentration of cytotoxicagents or radionuclides in tumors and should lessen the toxicity tonormal tissues.

In a preferred embodiment of the present invention the antibody is usedfor a treatment of cancers or tumors, preferably neuroblastoma,glioblastoma, rhabdomyosarcoma, breast carcinoma, melanoma, pancreaticcancer, B-cell non-Hodgkin's lymphoma, thyroid carcinoma, small celllung carcinoma, retinoblastoma, Ewing sarcoma, prostate cancer, coloncancer, or pancreatic cancer, preferably glioblastoma, neuroblastoma andrhabdomyosarcoma. ALK expression and protein has been detected in manysoft tissue tumors (Li et al., 2004). Full-length ALK has been found inthese human tumors. Furthermore, the antibody is preferably used forlocal treatments. Most preferred is local treatment of glioblastoma.

Another aspect of the present invention is to provide a DNA sequenceencoding the antibody of the present invention. A suitable prokaryoticexpression vector for ESBA512 (SEQ. ID. No: 19) is pTFT74 (see SEQ. ID.No: 90 for the sequence including the ESBA512 coding sequence). Therein,the ESBA512 coding sequence is under the control of the T7-promotor andthe recombinant gene product is usually purified over inclusion bodies.Another preferred prokaryotic expression vector is pAK400, wherein theESBA512 sequence is his-tagged for simplified purification (see SEQ. ID.No: 89 for the sequence including the ESBA512 coding sequence). The geneproduct is secreted by the host cell into the periplasm.

In addition, an expression vector comprising said DNA sequence and asuitable host cell transformed with said expression vector is provided.Preferably, said host cell is an E. coli cell.

Yet another aspect of the present invention is the production of theantibody of the present invention, comprising culturing the host cellthat is transformed with the expression vector for said antibody, underconditions that allow the synthesis of said antibody and recovering itfrom said culture.

Another aspect of the present invention is to provide an ALK epitope,comprising or consisting essentially of residues 391-406 of SEQ IDNO: 1. Said epitope is suitable for identifying, screening, or raisinganti-ALK antibodies or fragments thereof. Preferably, the antibody orantigen binding fragment thereof that is capable of specifically bindingresidues 391-406 (SEQ ID NO:1) of an isolated ALK protein or fragmentthereof. More preferably, the antibody is a single chain antibody(scFv), Fab fragment, IgG, or IgM.

In a further aspect, an ALK vaccine comprising an isolated ALK proteinor a fragment thereof, or a nucleic acid encoding an epitope of ALK isprovided. Preferably, the vaccine comprises residues 391-406 of anisolated ALK protein. Said vaccine is preferably formulated with acarrier, adjuvant, and/or hapten to enhance the immune response.

The sequences of the present invention are the following ones:

SEQ.ID. No. 1: GRIGRPDNPFRVALEY Human ALK epitope peptide (amino acids391-406 of the ALK protein); underlined residues are differ- ent in themouse homologue. SEQ.ID. No. 2: RDAWLDVLSDGFDY ESBA521 CDR 3 of VH.Residues obtained after randomization are underlined. SEQ.ID. No. 3:ATWDNDKWGVV ESBA521 CDR 3 of VL. Residues obtained after randomizationare underlined. SEQ.ID. No. 4:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAWLDVLSDGFDYWGQGTLVTVSS VH of ESBA521. CDRs are underlined. SEQ.ID. No.5: QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDNDKWGVV FGGGTKLEVLG VL ofESBA521. CDRs are underlined. SEQ.ID. No. 6: AISGSGGSTYYADSVKG VH CDR 2of ESBA521 SEQ.ID. No. 7: AINMKGNDRYYADSVGK VH CDR 2 of scFv 265.1SEQ.ID. No. 8: AIRTNSKEYYADSVKG VH CDR 2 of scFv 43.2 SEQ.ID. No. 9AIKTDGNHKYYADSVKG VH CDR 2 of scFv 100.2 SEQ.ID. No. 10: RTDSKEQYYADSVKGVH CDR 2 of scFv 2.11 SEQ.ID. No. 11: ETSSGSTYYADSVKG VH CDR 2 of scFv28.11 SEQ.ID. No. 12: NTGGGSTYYADSVKG VH CDR 2 of scFv 33.11 SEQ.ID. No.13: NTRGQNEYYADSVKG VH CDR 2 of scFv 4.12 SEQ.ID. No. 16GGGGSGGGGSGGGGSSGGGS Linker connecting VL and VH SEQ.ID. No. 17 YYADSVKGC-terminal half of CDR2 of ESBA521 and its derivatives. SEQ.ID. No. 18DAGIAVAGTGFDY VH CDR3 of FW4.4 SEQ.ID. No. 19QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDNDKWGVVFGGGTKLEVLGGGGGSGGGGSGGGGSSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAWLDVLSDGFDYWGQGTLVT VSS scFv ESBA521,CDRs underlined, linker in italics SEQ. ID. No. 20QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVVFGGGTKLTVLGGGGGSGGGGSGGGGSSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGIAVAGTGFDYWGQGTLVT VSS FW4.4, CDRs areunderlined, linker in italics SEQ ID No. 21cagtctgtgctgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctgctccggaagcacctccaacattggcgataattatgtatcctggtaccaacaactcccaggaacagccccccaactcctcatttatgacaatactaaacgaccctcagggattcctgaccggttctctggctccaagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattattactgcgcgacctgggataatgataagtggggtgtggttttcggcggagggaccaagctcgaggtcc-taggt Nucleic acid sequence of ESBA521 VLCDRs are underlined SEQ ID No. 22gaggtgcagctggtggagtccgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagCtgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgcgcgcgtgatgcgtggttggatgtgctttcggatggctttgactactggggccagggaaccct ggtcaccgtctcctcgNucleic acid sequence of ESBA521 VH CDRs are underlined SEQ ID No. 23cagtctgtgctgacgcagccgccctcagtgtctgcggccccaggacagaaggtcaccatctcctgctccggaagcacctccaacattggcgataattatgtatcctggtaccaacaactcccaggaacagccccccaactcctcatttatgacaatactaaacgaccctcagggattcctgaccggttctctggctccaagtctggcacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattattactgcgcgacctgggataatgataagtggggtgtggttttcggcggagggaccaagctcgaggtcctaggtggtggtggtggttctggtggtggtggttctggcggcggcggctccagtggtggtggatccgaggtgcagctggtggagtccgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgcgcgcgtgatgcgtggttggatgtgctttcggatggctttgactactggggccagggaaccctggtcacc gtctcctcg Nucleicacid sequence of ESBA521 CDRs are underlined, linker in italics

The invention is now further described by means of examples:

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques in polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning Cold Spring Harbor Laboratory Press (1989); Antibody EngineeringProtocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr(1996); Antibody Engineering: A Practical Approach (Practical ApproachSeries, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A LaboratoryManual, Harlow et al., C.S.H.L. Press, Pub. (1999); and CurrentProtocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons(1992).

Experiment 1: Screening to Identify Alk-Binding scFvs

In a wealth of structural studies on antibody-antigen interactions itwas found that residues in the complementarity-determining region 3(CDR-H3) of the heavy chain generally contribute the most substantialcontacts to the antigen (Chothia and Lesk, 1987; Chothia et al., 1985;Padlan, 1994). We applied our recently described yeast two-hybridantigen-antibody interaction screening technology to directly isolateantigen-binding scFvs by screening of four scFv libraries of randomizedsynthetic CDR-H3 sequences (Auf der Maur et al., 2002). The fourlibraries are based on four different stable human scFv frameworks inwhich 7 amino acids within the third CDR loop of the variable heavychain (VH-CDR3) were randomized. The randomized parts were introduced bystandard PCR cloning techniques. The scFv libraries were screenedagainst a 16 amino acid peptide derived from the extracellular domain ofthe human tyrosine receptor kinase Anaplastic Lymphoma Kinase (ALK) by ayeast screening system called “Quality Control” (Auf der Maur et al.,2001; Auf der Maur et al., 2004). Briefly, the Quality Controltechnology is an antigen-independent intrabody selection system foridentifying from a natural pool of human variable-light (VL) andvariable-heavy (VH) chains those VL and VH combinations with favourablebiophysical properties, such as stability, solubility and expressionyield. One promising and specific binder from one of the four scFvlibraries was isolated after the first screening round. This particularscFv was derived from the framework FW4.4 library. FW4.4 (SEQ. ID. No.20) consists of a VL domain (lambda1) connected by a classical flexibleglycine-serine linker (GGGGS)₄ to a VH₃ domain. The VH CDR3 of FW4.4comprises 13 amino acids (DAGIAVAGTGFDY; SEQ. ID. No. 18). To constructthe library, the central part of the VH CDR3 (DAXXXXXXXGFDY) wasrandomized by standard PCR-cloning methods using a degeneratedoligonucleotide. The last two residues (Asp and Tyr) were kept constant,because their structural importance was demonstrated in many cases(Chothia and Lesk, 1987). The remaining residues were not modified inorder to keep the complexity of the library in manageable dimensions.The scFv library was cloned in a yeast expression vector (pLib1) asC-terminal fusion to the transcriptional activation domain of Gal4 (Aufder Maur et al., 2002).

The ligand-binding domain (LBD) of human Alk was chosen as antigen forthe interaction screen (Stoica et al., 2001). This 16 amino acid peptidewas cloned into another yeast expression vector (pBait1) as C-terminalfusion to the DNA-binding protein LexA.

The reporter yeast strain YDE173 (Auf der Maur et al., 2002) containingthe stably integrated reporter genes HIS3 and lacZ under the control ofsix LexA-binding sites was transformed with the bait vector expressingthe Alk LBD fused to LexA together with the random CDR-H3 scFv libraryfused to the Gal4 activation domain. Transformed cells were selected onplates lacking histidine and containing 2.5 mM 3-amino-triazole (3-AT),which is a competitive inhibitor of the HIS3 gene product. Growingcolonies were picked over a period of six days and the library plasmidswere isolated. The same reporter strain was transformed with the rescuedplasmids to confirm antigen-dependent gene activation. A quantitativeliquid β-galactosidase assay was performed to measure binding-strengthbetween the Alk LBD, i.e. the 16 amino acid ALK peptide, and theselected scFv. The scFv with highest reporter gene activation alsodemonstrated best affinity (−31 nM) for the Alk LBD peptide in ELISA(data not shown).

The sequences of other VH CDR3 sequences identified as contributing toALK binding are provided below in Table 1a.

clone VH CDR 3 (mutated residues) WT FW4.4 DAGIAVAGTGFDY (SEQ. ID. No.24) H5 SH 2.1 DAKFMSDGIGFDY (SEQ. ID. No. 25) H5 SH 4.1 DAWGWTILSGFDY(SEQ. ID. No. 26) H5 SH 5.1 DAAYMIRYEGFDY (SEQ. ID. No. 27) H5 SH 2.3DAWIYWAREGFDY (SEQ. ID. No. 28) H5 SH 3.3 DACMTYSREGFDY (SEQ. ID. No.29) H5 SH 5.3 DAWLDVLSDGFDY (SEQ. ID. No. 30) H5 SH 14.3 DAPSVNDREGFDY(SEQ. ID. No. 31)

The sequences of other suitable frameworks are provided below in Table1b.

FW Sequence 5.2 EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPRLLIYDTSKRATGTPARFSGSGSGTDFTLTISSLEPEDSALYYCQQRNSWPHTFGGGTKLEIKRGGGGGSGGGGSGGGGSSGGGSEVQLVESGGGVAQPGGSLRVSCAASGFSFSSYAMQWVRQAPGKGLEWVAVISNDGRIEHYADAVRGRFTISRDNSQNTVFLQMNSLRSDDTALYYCAREIGATGYL DNWGQGTLVTVSS (SEQ. ID.No. 15)

Experiment 2: Affinity Maturation

In order to obtain an scFv with higher affinity, this primary binder wassubjected to a further affinity maturation process by mutagenesis and asecond screening round in yeast. Enabling affinity maturation, theexpression level of the LexA Alk LBD peptide fusion protein was reducedin order to lessen reporter gene activation driven by the interaction ofthe primary binder with the Alk LBD peptide. The strong actin promoteron the pBait1 vector was exchanged with the truncated and thus lessactive version of the ADH promoter (alcohol dehydrogenase) resulting inpBait3. This reduction of the bait expression level, in the presence ofthe primary binder, was sufficient to inhibit growth on plates lackinghistidine and containing 5 mM 3-AT. Mutagenesis of the primary binderfor affinity maturation was accomplished by randomizing parts of theCDR3 within the variable light chain. This was performed directly inyeast by homologous recombination (Schaerer-Brodbeck and Barberis,2004). The VL CDR3 of FW4.4 comprises 11 amino acids (SEQ. ID. No. 14:GTWDSSLSGVV). The first two positions were partially randomized, suchthat the first position either encodes Gly, Ala or Gln, and the secondposition Thr, Ser or Ala. At the positions 5 to 8 within VL CDR3 allamino acid residues were allowed. The remaining positions were keptconstant. Randomization was introduced by PCR. The resulting PCR producthad a size of 356 bp and comprised the randomized CDR cassette with 267bp upstream and 27 bp downstream framework sequences. This product isthe so-called donor PCR fragment, which bears homologies to the targetvector. The target vector is the yeast plasmid (pLib1) encoding theprimary binder fused to the activation domain of Gal4. In order toimprove efficiency of homologous recombination and to exclude falsepositives in the subsequent screening, the CDR-L3 in the target vectorwas slightly modified. A unique SacI restriction site was introduced inthe middle of VL CDR3, which leads to a frameshift in the scFv encodingpart of the fusion protein and results in a truncated protein unable tobind to the Alk LBD. In addition, the SacI site enables linearization ofthe target vector, which enhances recombination efficiency in yeast.

The screening was launched by pre-transformation of the reporter yeaststrain YDE173 with the plasmid (pBait3) expressing the LexA Alk LBD fromthe truncated ADH promoter. This pre-transformed yeast cells were madecompetent again and co-transformed with the linearized target vector andwith the donor PCR fragment, which bears homologies upstream anddownstream of the VL CDR3. Upon homologous recombination between the PCRproduct and the target vector, the novel VL CDR3 sequence is integratedinto the corresponding site of the target vector. As a net result ofthis event the primordial VL CDR3 gets exchanged with the randomized VLCDR3. This allows reconstitution of a circular plasmid that expresses afully functional fusion protein with a novel VL CDR3 sequence, whichwill activate reporter gene expression and enable growth on selectiveplates upon interaction with the Alk LBD peptide.

A total of 119 clones grew on selective plates over an observationperiod of 6 days. These clones were picked and the library plasmids wereisolated and retransformed into the same reporter yeast strain. Aquantitative liquid β-galactosidase assay was performed to measurebinding strength between the Alk LBD (antigen) and the affinity-maturedscFv. 20 clones with highest lacZ activation were also tested in ELISAwith Alk LBD peptide. The best candidate revealed a K_(D) of about 7 nM(FIG. 3) and was named ESBA521.

The sequences of other VL CDR3 sequences identified as contributing toALK binding are provided below in Table 1c.

clone VL CDR 3 WT FW4.4 GTWDSSLSGVV (SEQ. ID. No. 32)  5.3-9.1AAWDSVKHGVV (SEQ. ID. No. 33)  5.3-21.1 AAWDNSMRGVV (SEQ. ID. No. 34) 5.3-22.1 AAWDTMRYGVV (SEQ. ID. No. 35  5.3-25.1 AAWDTTRVGVV (SEQ. ID.No. 36)  5.3-27.1 ASWDTMLKGVV (SEQ. ID. No. 37)  5.3-28.1 ASWDTPTCGVV(SEQ. ID. No. 38)  5.3-29.1 ATWDISRCGVV (SEQ. ID. No. 39)  5.3-46.1ATWDTVCAGVV (SEQ. ID. No. 40)  5.3-53.1 ATWDVDVFGVV (SEQ. ID. No. 41) 5.3-57.1 ATWDDVVGGVV (SEQ. ID. No. 42)  5.3-86.1 AAWDSFYNGVV (SEQ. ID.No. 43)  5.3-94.1 ASWDTLIEGVV (SEQ. ID. No. 44)  5.3-107.1 ATWDNDKWGVV(SEQ. ID. No. 45)  5.3-112.1 AAWDSTTCGVV (SEQ. ID. No. 46)  5.3-113.1ATWDMWMKGVV (SEQ. ID. No. 47)  5.3-117.1 GTWDSSLSGVV (SEQ. ID. No. 48) 5.3-118.1 AAWDWVLGGVV (SEQ. ID. No. 49) 14.3-6.1 ATWDNPGQGVV (SEQ. ID.No. 50) 14.3-7.1 ATWDDWVIGVV (SEQ. ID. No. 51) 14.3-8.1 ASWDDQKWGVV(SEQ. ID. No. 52) 14.3-9.1 ATWDTNRHGVV (SEQ. ID. No. 53) 14.3-12.1ASWDDLHIGVV (SEQ. ID. No. 54) 14.3-13.1 ASWDEEAWGVV (SEQ. ID. No. 55)14.3-21.1 ATWDYIKIGVV (SEQ. ID. No. 56) 14.3-48.1 ATWDTFERGVV (SEQ. ID.No. 57) 14.3-49.1 ATWDSNLIGVV (SEQ. ID. No. 58)  5.3-24.,1 ATWDNNTCGVV(SEQ. ID. No. 59)  5.3-3.1 AAWDCDINGVV (SEQ. ID. No. 60)  5.3-8.1ASWDSMKIGVV (SEQ. ID. No. 61)  5.3-19.1 ATWDCTRAGVV (SEQ. ID. No. 62)

Experiment 3: the ScFv ESBA521 Specifically Binds to the TransmembraneForm of Human ALK

In order to test whether the newly identified scFv was able to alsorecognize the transmembrane human ALK protein on the surface of livingcells, immunostaining experiments of transiently transfected HELA cellswere performed. ESBA521 reacts with the ALK protein in a comparable wayas a polyclonal antibody (FIG. 4). In a control experiment it was shownthat the framework 4.4 scFv does not react with human ALK. Surprisingly,ESBA521 only binds to the human Alk protein, but not to thecorresponding mouse protein, although the mouse antigenic peptide onlydiffers in two amino acid positions from the human peptide sequence. Bycontrast, the polyclonal ALK antibody recognizes both human and mouseprotein. Therefore, binding of ESBA521 is specific for the human ALKprotein at the cell surface.

Experiment 4: Isolation of Improved Binders by PCR Mutagenesis of VH CDR2

To further improve antigen binding, ESBA521 was used as the startingscFv in a further round of affinity maturation using the same two-hybridapproach as described for the first round of affinity maturation, exceptin this case CDR2 of VH was changed by PCR mutagenesis and transformedinto the yeast recipient, wherein homologous recombination at CDR2 isenforced in analogous way. Again, a restriction site was introduced inCDR2 to enable linearization of the target plasmid. The mutationsintroduced in CDR2 are given in Table 2.

scFv (performing best VH CDR 2 (mutated residues) WT (ESBA521)ATSGSGGSTYYADSVKG (SEQ. ID. No. 63)   1.1 AI-KTDGQNYYADSVKG (SEQ. ID.No. 64)  17.1 AIRSDGNERYYADSVKG (SEQ. ID. No. 65)  35.1AINTNGNEKYYADSVKG (SEQ. ID. No. 66)  64.1 AISTNGKERYYADSVKG (SEQ. ID.No. 67) 130.1 AIRTQSQEEYYADSVKG (SEQ. ID. No. 68) 152.1AIKSRSQEQYYADSVKG (SEQ. ID. No. 69) 167.1 AIKSHSQQQYYADSVKG (SEQ. ID.No. 70) 214.1 AINSEGQQRYYADSVKG (SEQ. ID. No. 71) 225.1AIKSKGQNKYYADSVKG (SEQ. ID. No. 72) 262.1 AIRTNSEEKYYADSVKG (SEQ. ID.No. 73) 265.1 AINMKGNDRYYADSVKG (SEQ. ID. No. 74)  43.2AI-RTNSKEYYADSVKG (SEQ. ID. No. 75)  70.2 AIKTESQQRYYADSVKG (SEQ. ID.No. 76)  99.2 AINSNGKQDYYADSVKG (SEQ. ID. No. 77) 100.2AIKTDGNHKYYADSVKG (SEQ. ID. No. 78) 109.2 AIDTKGNGQYYADSVKG (SEQ. ID.No. 79) 146.2 AIRSDSSHKYYADSVKG (SEQ. ID. No. 80) 173.2AINTKSNEQYYADSVKG (SEQ. ID. No. 81) 199.2 AIRTDSKNSYYADSVKG (SEQ. ID.No. 82)   2.11 AIRTDSKEQYYADSVKG (SEQ. ID. No. 83)  19.11AIRTNSKEEYYADSVKG (SEQ. ID. No. 84)  28.11 AIETSSGSTYYADSVKG (SEQ. ID.No. 85)  33.11 AINTGGGSTYYADSVKG (SEQ. ID. No. 86)   4.12AINTRGQNEYYADSVKG (SEQ. ID. No. 87)   6.12 AISTSG-STYYADSVKG (SEQ. ID.No. 88)

Among the isolated scFvs obtained after this procedure, seven turned outto have significantly improved affinity with a K_(d) in the range of 2-3nM (FIG. 5), the best of them being 28.11.

Experiment 5: Prevention of Tumor-Growth Upon Administration of Anti-ALKAntibody

The progenitors of the antibody ESBA521 were selected to bind to aminoacids 391-406 of ALK, which comprises the amino acids (396-406) that areknown to bind pleiotrophin (Stoica 2001). ESBA521 was obtained byrandomizing additional amino acids in the CDRs of its progenitor and byselecting for binders that bind to the 391-406 amino acid stretchcontained in its natural context of the ALK extracellular domain (ECD).These proceedings resulted in an antibody, which binds the ALK ECD withhigh affinity at the same site that binds PTN. To our knowledge. This isthe first monoclonal antibody that specifically targets the PTN bindingsite of ALK. Accordingly, ESBA521 is predicted to have high affinity tothe ALK ECD and efficiently competes with pleiotrophin (PTN) and midkine(MK) for binding to the ALK receptor, and thus, the ESBA521 antibody issuitable for inhibiting both MK and PTN ligand binding to the ALKprotein.

Because ALK and its ligands are involved in neoplasia, tumor invasionand angiogenesis, inhibition of the interaction between ALK and itscognate ligands disrupts ALK mediated tumorgenesis.

The effect of ESBA521 on a specific tumor can be determined by thefollowing two assays described below.

In a first assay, xenograft experiments are prepared in order todetermine the cancer growth rate-limiting role of ALK (Powers 2002).Briefly, a U87MG cell suspension of 20 million cells/ml mediasupplemented with 10% fetal calf serum are prepared. These are injectedinto NU/NU mice and resultant tumors are measured. Test antibodies,preferably full length antibodies, more preferably, pegylatedantibodies, are introduced and tumor growth is assessed.

While there are shown and described presently preferred embodiments ofthe invention, it is to be distinctly understood that the invention isnot limited thereto but may be otherwise variously embodied andpracticed within the scope of the following claims.

REFERENCES

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1-40. (canceled)
 41. An isolated antibody which binds the human ALKprotein, said antibody comprising a variable heavy chain CDR3 of asequence with at least 50% sequence identity to the sequence SEQ. ID.NO.
 2. 42. The antibody of claim 41, comprising a variable heavy chainCDR3 of a sequence with at least 60% sequence identity to the sequenceSEQ. ID. NO.
 2. 43. The antibody of claim 41, comprising a variableheavy chain CDR3 of a sequence with at least 70% sequence identity tothe sequence SEQ. ID. NO.
 2. 44. The antibody of claim 41, comprising avariable heavy chain CDR3 of a sequence with at least 75% sequenceidentity to the sequence SEQ. ID. NO.
 2. 45. The antibody of claim 41,comprising a variable heavy chain CDR3 of a sequence with at least 85%sequence identity to the sequence SEQ. ID. NO.
 2. 46. The antibody ofclaim 41, comprising a variable heavy chain CDR3 of a sequence with atleast 90% sequence identity to the sequence SEQ. ID. NO.
 2. 47. Theantibody of claim 1, comprising a variable heavy chain CDR3 of thesequence SEQ. ID. NO.
 2. 48. The antibody of claim 1, wherein bindingthe human ALK protein is specific.
 49. The antibody of claim 1, which isan antibody fragment, scFv antibody or a Fab fragment.
 50. The antibodyof claim 1, which is an scFv antibody.
 51. The antibody of claim 1,comprising a H3 VH domain and a lambda1 VL domain.
 52. The antibody ofclaim 1, wherein the framework of VH and VL is stable and soluble so asto be functional in an intracellular reducing environment.
 53. Theantibody of claim 1, comprising the framework 4.4.
 54. The antibody ofclaim 1, said antibody binding a 16-amino acid ALK epitope peptide of asequence of at least 75% sequence identity to the sequence SEQ. ID.NO.
 1. 55. The antibody of claim 54, wherein the ALK epitope peptide hasthe sequence SEQ. ID. NO
 1. 56. The antibody of claim 54, whose affinityfor the ALK epitope peptide is characterized by a K_(d) of 30 nM orless.
 57. The antibody of claim 54, whose affinity for the ALK epitopepeptide is characterized by a K_(d) of 10 nM or less.
 58. The antibodyof claim 54, whose affinity for the ALK epitope peptide is characterizedby a K_(d) of less than 3 nM.
 59. The antibody of claim 1, comprising avariable light chain CDR3 of a sequence with at least 50% sequenceidentity to the sequence SEQ. ID. NO.
 3. 60. The antibody of claim 59,comprising a variable light chain CDR3 of a sequence with at least 60%sequence identity to the sequence SEQ. ID. NO.
 3. 61. The antibody ofclaim 59, comprising a variable light chain CDR3 of a sequence with atleast 70% sequence identity to the sequence SEQ. ID. NO.
 3. 62. Theantibody of claim 59, comprising a variable light chain CDR3 of asequence with at least 80% sequence identity to the sequence SEQ. ID.NO.
 3. 63. The antibody of claim 59, comprising a variable light chainCDR3 of a sequence with at least 85% sequence identity to the sequenceSEQ. ID. NO.
 3. 64. The antibody of claim 59, comprising a variablelight chain CDR3 of a sequence with at least 90% sequence identity tothe sequence SEQ. ID. NO.
 3. 65. The antibody of claim 59, comprising avariable light chain CDR3 of the sequence SEQ. ID. No.
 3. 66. Theantibody of claim 1, said antibody comprising a VH sequence of SEQ. ID.NO. 4 and a VL sequence of SEQ. ID. NO.
 5. 67. The antibody of claim 1,comprising at least one mutation in at least one of the CDRs resultingin a higher affinity characterized by a K_(d) of less than about 3 nM.68. The antibody of claim 67, wherein said at least one mutation is inCDR1 or CDR2 of VH or VL.
 69. The antibody of claim 67, wherein said atleast one mutation is in CDR2 of VH.
 70. The antibody of claim 1,comprising a variable heavy chain CDR2 comprising a sequence selectedfrom the group of SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ.ID. NO. 10, SEQ. ID. NO. 11, SEQ. ID. NO. 12, or SEQ. ID. NO.
 13. 71.The antibody of claim 70, wherein said variable heavy chain CDR2sequence is preceded by amino acids residues alanine-isoleucine andfollowed by the sequence of SEQ. ID. NO.
 17. 72. The antibody of claim1, said antibody comprising a VH sequence of SEQ. ID. NO. 14 and a VLsequence of SEQ. ID. NO.
 15. 73. The antibody of claim 50, said antibodycomprising the structure NH₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH,wherein the linker has the sequence SEQ. ID. N).
 16. 74. The antibody ofclaim 1, said antibody being radiolabeled or toxin-labeled.
 75. A methodfor the treatment of cancers or tumors comprising administering to asubject the antibody of claim
 1. 76. The method of claim 75, wherein theantibody inhibits binding of MK or PTN to ALK.
 77. The method of claim75, wherein the antibody inhibits ALK-mediated signaling.
 78. The methodof claim 75, wherein the antibody is administered in combination with ananticancer agent.
 79. The method of claim 78, wherein the anticanceragent is methotrexate.
 80. The method of claim 75, wherein the treatmentis a treatment of neuroblastoma, glioblastoma, rhabdomyosarcoma, breastcarcinoma, melanoma, pancreatic cancer, B-cell NHL, thyroid carcinoma,small cell lung carcinoma, retinoblastoma, ewing sarcoma, prostatecancer, colon cancer, pancreatic cancer, lipoma, liposarcoma orfibrosarcoma.
 81. An isolated DNA molecule encoding the antibody ofclaim
 1. 82. An expression vector comprising the DNA molecule of claim81.
 83. A host cell transformed with the expression vector of claim 82.84. The host cell of claim 83, which is an E. Coli cell.
 85. A methodfor the production of an antibody comprising culturing the host cell ofclaim 84 under conditions that allow for the synthesis of said antibodyand recovering said antibody.
 86. An ALK vaccine comprising an isolatedALK protein or a fragment thereof.
 87. The vaccine of claim 86, whereinthe vaccine comprises residues 391-406 of an isolated ALK protein. 88.The vaccine of claim 86, wherein the vaccine consists of residues391-406 of an isolated ALK protein formulated with a carrier, adjuvantor hapten to enhance the immune response to the ALK protein or fragmentthereof.
 89. An ALK vaccine comprising an isolated nucleic acid encodingan epitope of ALK.
 90. An isolated antibody, or antigen binding fragmentthereof, that specifically binds residues of a region spanning aminoacid residues 391±3 and 406±3 (SEQ. ID. No: 91) of an ALK protein. 91.The antibody of claim 90, which specifically binds amino acids 391-406(SEQ. ID. No: 1) of an ALK protein.
 92. The antibody of claim 90,wherein the antibody is a single chain antibody (scFv), Fab fragment,IgG, or IgM.
 93. An isolated ALK epitope suitable for identifying,screening, or raising anti-ALK antibodies or fragments thereof, whereinthe ALK epitope is a fragment of a region spanning amino acid residues391±3 and 406±3 (SEQ. ID No: 91) of an ALK protein.
 94. The epitope ofclaim 93, wherein the ALK epitope is a fragment of a region spanningamino acids 391-406 (SEQ. ID. No: 1) of an ALK protein.
 95. An isolatedALK epitope suitable for identifying, screening, or raising anti-ALKantibodies or fragments thereof, wherein the ALK epitope comprises aregion spanning amino acid residues 391±3 and 406±3 (SEQ. ID No: 91) ofan ALK protein.
 96. The epitope of claim 95, wherein the ALK epitopecomprises a region spanning amino acids 391-406 (SEQ. ID. No: 1) of anALK protein.
 97. The epitope of claim 95, wherein the ALK epitopeconsists essentially of a region spanning amino acid residues 391±3 and406±3 (SEQ. ID No: 91) of an ALK protein.
 98. The epitope of claim 95,wherein the ALK epitope consists essentially of a region spanning aminoacids 391-406 (SEQ. ID. No: 1) of an ALK protein.
 99. The antibody ofclaim 1, wherein said antibody binds an epitope that is a fragment of aregion spanning amino acid residues 391±3 and 406±3 (SEQ. ID No: 91) ofan ALK protein.
 100. The antibody of claim 99, wherein the ALK epitopeis a fragment of a region spanning amino acids 391-406 (SEQ. ID. No: 1)of an ALK protein.
 101. The antibody of claim 1, wherein said antibodybinds an epitope comprising a region spanning amino acid residues 391±3and 406±3 (SEQ. ID No: 91) of an ALK protein.
 102. The antibody of claim101, wherein the ALK epitope comprises a region spanning amino acids391-406 (SEQ. ID. No: 1) of an ALK protein.
 103. The antibody of claim1, wherein said antibody binds an epitope consisting essentially of aregion spanning amino acid residues 391±3 and 406±3 (SEQ. ID No: 91) ofan ALK protein.
 104. The antibody of claim 103, wherein the ALK epitopeconsists essentially of a region spanning amino acids 391-406 (SEQ. ID.No: 1) of an ALK protein.